Compositions and methods of use for anti-inflammatory agents

ABSTRACT

This invention relates to the use of a phosphorylcholine-containing glycoprotein in the treatment or prophylaxis of diseases associated with inflammation. In particular, the present invention relates to the use of ES-62 for the treatment or prophylaxis of autoimmune diseases associated with abnormal inflammation, such as rheumatoid arthritis.

FIELD OF THE INVENTION

The present invention relates to the use of a glyco-protein for thetreatment or prophylaxis of diseases associated with inflammation, inparticular the present invention relates to the use of ES-62 for thetreatment or prophylaxis of autoimmune diseases associated with abnormalinflammation, such as rheumatoid arthritis (RA).

BACKGROUND OF THE INVENTION

Filarial nematodes are arthropod-transmitted parasites of vertebratesincluding humans. Infection is long-term with individual worms survivingfor in excess of five years. The consensus of opinion amongst workers inthis field is that such longevity reflects suppression or modulation ofthe host immune system and indeed “defects” in immune responsivenesshave been revealed in infected individuals. These defects incorporateimpairment of lymphocyte proliferation and bias in production of bothcytokines and antibodies. With respect to cytokines the bias can beshown in reduced production of the pro-inflammatory IFN-γ and increasedproduction of the anti-inflammatory IL-10. For antibodies, there areimbalances in IgG subclasses—greatly elevated IgG4 (an antibody oflittle value in eliminating pathogens due to an inability to activatecomplement or bind with high affinity to phagocytic cells); decreases inother IgG subclasses. Overall therefore, the picture is of an immuneresponse demonstrating a somewhat suppressed, anti-inflammatoryphenotype which is often classified as “TH-2” (“TH” is derived from acategory of T-lymphocyte referred to as “helper”). It has beenspeculated that such a phenotype is conducive to both parasite survivaland host health, the latter by limiting pathology resulting from anover-aggressive immune response. Consistent with this, it is noteworthythat the majority of humans who harbour these parasites demonstratelittle evidence of detrimental pathology.

Modulation of the host immune system is likely to involve the activeparticipation of filarial nematodes and hence considerable effort hasbeen spent in characterising the biological properties of moleculessecreted by the worms.

Goodridge et al. in the Journal of Immunology, 2001, 167, discussmodulation of macrophage cytokine production by ES-62 resulting insuppression of the production of pro-inflammatory cytokines which maycontribute to the immunomodulatory properties of ES-62 that drive thegeneration of immune responses with an anti-inflammatory and/or TH-2phenotype. Although this article provides a rationale for the changesseen in the immune response, no guidance is provided on whether ES-62may be useful in the clinic for preventing or treating any particulardisease(s), in particular those diseases involving inflammation.

Harnett and Harnett in Biochemica et Biophysica Acta, 1539, (2001), 7–15report investigations into the underlying mechanism of action of ES-62and phosphorylcholine (PC). It is concluded that PC has various actions,including a number of immunomodulatory properties.

The prior art, at best, may only suggest that ES-62 has a role in themediation of diseases involving an inflammatory response due to theeffect of ES-62 on production of inflammatory cytokines. However, theseinvestigations have been performed only in vitro, and no evidence ispresented to show that ES-62 may be used to treat disorders associatedwith inflammation.

Evidence of such molecules having a clinical use has hitherto not beendemonstrated, and thus an object of the present invention is to obviateand/or mitigate the current treatment inadequacies of diseasesassociated with inflammation.

SUMMARY OF THE INVENTION

Broadly speaking the inventors have been generally investigating theES-62 molecule which appears to have an immunomodulatory effect.

According to a first aspect of the present invention there is providedthe compound ES-62 or physiologically active derivatives for use as amedicament.

The medicament may be used in the in vivo treatment or prophylaxis ofinflammatory diseases in an animal. The animal requiring treatment orprophylaxis is usually a human or non-human mammal.

According to a second aspect of the present invention, there is providedthe compound ES-62 or physiologically active derivatives thereof for usein treatment or prophylaxis of inflammatory diseases in an animal.

The animal requiring treatment or prophylaxis is usually a human ornon-human mammal.

Many diseases involve inflammation, and particularly relevant diseasesfor treatment or prophylaxis according to the present invention areautoimmune diseases such as type 1 diabetes melitus, rheumatoidarthritis, psoriasis, multiple sclerosis, autoimmune hepatitis,sarcoidosis, inflammatory bowel disease, and chronic obstructivepulmonary disease.

The present invention has particular application for the treatment orprophylaxis of rheumatoid arthritis.

According to a third aspect of the present invention there is providedthe use of ES-62 for the manufacture of a medicament for the treatmentor prophylaxis of inflammatory disease. In a fourth aspect of thepresent invention there is provided a method for the treatment orprophylaxis of inflammatory disease in an animal comprisingadministering to said animal a therapeutically or prophylacticallyeffective amount of ES-62 or physiologically functional derivativethereof.

Preferably, ES-62 is used therapeutically to treat inflammatory disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of ES-62 where potential N-linkedglycosylation sites are under-lined. Potential N-linked glycosylationsites are underlined. It is currently unknown as to how many of theseare utilized and of the utilized ones, how many have linkage withphosphorycholine-containing glycans.

FIG. 2 shows the phosphorylcholine-glycan component structure of ES-62(Haslam et al. (1997), Molecular and Biochemical Parasitology, 85,53).The numbers 2, 3, 4 and 6 refer to positions for the C-linkage. The +and − symbols merely mean that the units are optional. FIG. 2 also showsthat the phosphorycholine is bound to the glycan and not the protein.

FIG. 3 shows a summary of the experimental protocol for collagen-inducedarthritis.

FIG. 4 shows graphs indicating the development of arthritis with time.FIG. 4A. Mean articular index as a function of time. FIG. 4B. Incidence(%) of collagen-induced arthritis as a function of time. Data areexpressed as mean +/−SEM. PBS (n=13), ES-62 (n=12), ES-62 Multi (n=14).†p<0.05 PBS vs. ES-62, *p<0.05 PBS vs. ES-62 Multi, Mann-Whitney U-test.

FIG. 5 shows suppression of antigen-induced cell proliferation andpro-inflammatory cytokine production in lymph node cultures at day 33.FIG. 5A. Thymidine uptake (×10³ cpm) for collagen-treatment vs. controlsin PBS, ES-62, and ES-62 Multi conditions. FIG. 5B. IFN-γ (pg/ml) forcollagen-treatment vs. controls in PBS, ES-62, and ES-62 Multiconditions. FIG. 5C. TNF-α(pg/ml) for collagen-treatment vs. controls inPBS, ES-62, and ES-62 Multi conditions. FIG. 5D. IL-6 (pg/ml) forcollagen-treatment vs. controls in PBS, ES-62, and ES-62 Multiconditions. FIG. 5E. IL-10 (pg/ml) for collagen-treatment vs. controlsin PBS, ES-62, and ES-62 Multi conditions. Data are expressed as mean+/−S.D., *p<0.05.

FIG. 6 shows that ES-62 treatment maintains the inhibition of both cellproliferation and inflammatory cytokine production at day 50. FIG. 6A.Thymidine uptake (×10³ cpm) for collagen-treatment vs. controls in PBS,ES-62, and ES-62 Multi conditions. FIG. 6B. IFN-γ (pg/ml) forcollagen-treatment vs. controls in PBS, ES-62, and ES-62 Multiconditions. FIG. 6C. TNF-α (pg/ml) for collagen-treatment vs. controlsin PBS, ES-62, and ES-62 Multi conditions. FIG. 6D. IL-6 (pg/ml) forcollagen-treatment vs. controls in PBS, ES-62, and ES-62 Multiconditions. Data are expressed as mean +/−S.D., *p<0.05.

FIG. 7 shows a reduction in production of the TH-1 associated antibody,IG2a by ES-62 treatment at day 50. FIG. 7A. Detection levels ofanti-collagen IgG2a in PBS, ES-62, and ES-62 Multi conditions. FIG. 7B.Detection levels of anti-collagen IgG1 in PBS, ES-62, and ES-62 Multiconditions. Pooled data of individual measurements (n=5/group) &expressed as mean +/−SEM; *p<0.05, Mann-Whitney test.

FIG. 8 shows ES-62 treatment is effective in preventing proaression ofestablished arthritis. FIG. 8A. Mean articular index as a function oftime. FIG. 8B. Mean number of arthritic paws as a function of time. FIG.8C. Mean paw thickness (mm) as a function of time. Data are mean +/−SEM;PBS (n=10); ES-62 (sc 2 μg daily for 14 days, n=10). *p<0.05 **p<0.01Mann-Whitney test.

FIG. 9 shows ex-vivo suppression of antigen-induced cell proliferationin lymph node cultures upon ES-62 administration and also suppression ofproduction of certain pro-inflammatory cytokines in establishedcollagen-induced arthritis. FIG. 9A. Thymidine uptake (×10³ cpm) forcollagen-treatment vs. controls in PBS and ES-62 conditions. FIG. 9B.IFN-γ (pg/ml for collagen-treatment vs. controls in PBS and ES-62conditions. FIG. 9C. TNF-α (pg/ml) for collagen-treatment vs. controlsin in PBS and ES-62 conditions. FIG. 9D. IL-6 (pg/ml) forcollagen-treatment vs. controls in in PBS and ES-62 conditions. Data areexpressed as mean +/−SEM (n=5/group, d15 ES-62), *p<0.05 Student'sT-test.

FIG. 10 shows a reduction in production of the TH-1 antibody. IgG2a uponadministration of ES-62 in established collagen-induced arthritis. FIG.10A. Detection levels of anti-collagen IgG2a. FIG. 10B. Detection levelsof anti-collagen IgG1. Pooled data of individual measurements (n=5/groupd15 ES-62) & expressed as mean +/−SEM: *p<0.05, ANOVA test.

FIG. 11 shows sections of arthritic hind paws of mice stained withhematoxylin and eosin showing a prevention in cartilage surface erosionand inflammation in ES-62 treated mice. FIG. 11A. PBS-treated mice. FIG.11B. ES-62-treated mice. Mice were treated with PBS (FIG. 11A) or ES-62(FIG. 11B) according to the therapeutic protocol and arthritic hind pawswere removed and stained with hematoxylin and eosin. Profound cartilagesurface erosion and inflammation was observed in PBS controls.

FIG. 12 shows that ES-62 treatment has no effect on footpad incrementobserved in Leishmania mouse strains. FIG.12A. Mean footpad incremement(mm) as a function of time in PBS and ES-62 treatment conditions inBALB/c mice. FIG. 12B. Mean footpad incremement (mm) as a function oftime in PBS and ES-62 treatment conditions in C57/B6 mice (trial I).FIG. 12C. Mean footpad incremement (mm) as a function of time in PBS andES-62 treatment conditions in C57/B6 mice (trial II). FIG. 13 shows thatES-62 inhibits pro-inflammatory cytokine production from human synovialmembrane and fluid cells. FIG. 13A. TNFα release in single cellsuspensions prepared from synovial membranes: 1) with no treatment; 2)treated with LPS at 100 ng/ml or 1 μg/ml for 24 hours; or 3) treatedwith ES-62 and LPS at 100 ng/ml or 1 μg/ml for 24 hours. FIG. 13B. TNFαrelease in single cell suspensions prepared from synovial membranes: 1)with no treatment; 2) treated with LPS at 10 ng/ml for 24 hours; or 3)treated with ES-62 and LPS at 10 ng/ml for 24 hours.

DETAILED DESCRIPTION

The present inventors have given particular attention to the ES-62molecule, which is a glycoprotein with the unusual post-translationmodification of phosphorylcholine attachment to an N-type glycan. Theyfound this molecule to possess a plethora of immunomodulatory activitiesthat can be classified under the umbrella of “anti-inflammatory”. Thus,ES-62 reduces the ability of lymphocytes (both B- and T-) to proliferatein response to antigen, inhibits the ability of macrophages to producepro-inflammatory cytokines such as IL-12, TNF-α and IL-6, modulatesdendritic cell maturation to preferentially elicit TH-2-like responses,induces spleen cells to produce the anti-inflammatory cytokine, IL-10and biases antibody responses in a TH-2/anti-inflammatory direction.

ES-62 has been identified as a glycoprotein comprising the amino acidsequence shown in FIG. 1. The phosphorylcholine-glycan (PC-glycan)component of ES-62 is depicted as the structure shown in FIG. 2.

ES-62 may be prepared from spent culture medium from adult A. viteaemaintained in RPMI complete at 37° C. in an atmosphere of 5% CO₂/95% airand was passed through filtration units with 0.22 μm membranes (Sigma)to remove microfilariae. The medium was concentrated to a volume of 10ml/washed in PBS, pH 7.2, in a stirred cell with a PM10 membrane(Amicon, Upper Mill, UK). A further concentration step was undertakenusing Centricon tubes (Amicon) with a 100-kDa membrane (ES-62 has anative m.w. of 280 kDa) to give a final volume of 0.5 ml. This wasapplied to a 30×1 cm Superose 6 column (HR 10/30, Pharmacia), fitted toan isocratic FPLC system (Pharmacia), previously equilibrated with PBS,pH 7.2, at room temperature. The column was eluted at a flow rate of 0.5ml/mm and monitored for absorbance at 280 nm. The >95% of protein elutedas a single peak, which was confirmed to be ES-62 by analysis usingSDS-PAGE. This was concentrated to 2 mg/ml and stored at −20° C. in10-μl aliquots (Harnett, W. et al (1993) J. Immunol. 151. 4829).

Biochemical characterization of ES of adult A. viteae maintained inRPMI-1640 medium shows that it is dominated by one molecule, ES-62,so-called because it has a relative molecular mass of 62000 whenanalysed by SDS-PAGE under reducing conditions. It is estimated thatES-62 accounts for more than 95% of all of the protein released. ES-62is also actively secreted by A. viteae in vivo, as shown by itsdetection in the bloodstream of parasitized jirds. ES-62 is of relevanceto human filariasis because it shares considerable sequence homologywith recently characterized EST's of B. malayi and because it containsphosphorylcholine, a molecule found on ES of all human filarialnematodes examined to date.

Investigation of the mechanism of attachment of PC to ES-62 revealedthat it was via an N-type glycan (Harnett et al. (1993) ExperimentalParasitology. 77, 498, Houston & Harnett (1996), Journal ofParasitology, 82, 320). Structural analysis of the PC-glycan of ES-62indicated that at least 1–2 PC groups were present per glycan. Sequenceanalysis of ES-62 revealed that there were three potential N-linkedglycosylation sites in the mature protein (Harnett et al. (1999),Molecular and Biochemical Parasitology, 104, 11). Thus, each molecule ofES-62 also reveals two leucine rich repeat regions: one of theproperties of such motifs is that they promote protein dimerization andan estimation of the molecular mass of ES-62 by FPLC reveals a figure ofapproximately 280 000. Thus the native molecule appears to exist in atetrameric form.

Inflammation is the hallmark of many diseases but the prototypicalinflammatory diseases are the autoinimune diseases, which include type 1diabetes melitus, rheumatoid arthritis, psoriasis, multiple sclerosis,autoimmune hepatitis, sarcoidosis and inflammatory bowel disease. Suchchronic diseases are characteristically relapsing and remitting innature and current treatment is inadequate.

For use according to the present invention, the glyco-protein ES-62 ispreferably presented as a pharmaceutical formulation, comprising ES-62or other physiologically functional derivative thereof, together withone or more pharmaceutically acceptable carriers therefore andoptionally other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal and pulmonary administration e.g., by inhalation. The formulationmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association anactive compound with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of active compound. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an active compound with an inertliquid diluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activecompound, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmaimer. Cachets are analogous to capsules wherein an active compoundtogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active compound may also be formulated as dispersablegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet.

Formulations suitable for oral administration wherein the carrier is aliquid may be presented as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active compound is formulated in anappropriate release—controlling matrix, or is coated with a suitablerelease—controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active compound with the softenedor melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active compound inaqueous or oleaginous vehicles. Injectible preparations may be adaptedfor bolus injection or continuous infusion. Such preparations areconveniently presented in unit dose or multi-dose containers which aresealed after introduction of the formulation until required for use.Alternatively, an active compound may be in powder form which isconstituted with a suitable vehicle, such as sterile, pyrogen-freewater, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finelycomminuted powders which may conveniently be presented either in apierceable capsule, suitably of, for example, gelatin, for use in aninhalation device, or alternatively as a self-propelling formulationcomprising an active compound, a suitable liquid or gaseous propellantand optionally other ingredients such as a surfactant and/or a soliddiluent. Suitable liquid propellants include propane and thechlorofluorocarbons, and suitable gaseous propellants include carbondioxide. Self-propelling formulations may also be employed wherein anactive compound is dispensed in the form of droplets of solution orsuspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microliters, upon eachoperation thereof.

As a further possibility an active compound may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropnate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active compound in aqueous or oilysolution or suspension.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations described above may include,an appropriate one or more additional carrier ingredients such asdiluents, buffers, flavouring agents, binders, surface active agents,thickeners, lubricants, preservatives (including anti-oxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Therapeutic formulations for veterinary use may conveniently be ineither powder or liquid concentrate form. In accordance with standardveterinary formulation practice, conventional water soluble excipients,such as lactose or sucrose, may be incorporated in the powders toimprove their physical properties. Thus particularly suitable powders ofthis invention comprise 50 to 100% w/w and preferably 60 to 80% w/w ofthe active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/wof conventional veterinary excipients. These powders may either be addedto animal feedstuffs, for example by way of an intermediate premix, ordiluted in animal drinking water.

Liquid concentrates of this invention suitably contain ES-62 or a saltthereof and may optionally include a veterinarily acceptablewater-miscible solvent, for example polyethylene glycol, propyleneglycol, glycerol, glycerol formal or such a solvent mixed with up to 30%v/v of ethanol. The liquid concentrates may be administered to thedrinking water of animals.

EXPERIMENTAL Materials, Methods and Results

Eight-ten week old mice (DBA/1 strain) were given 2 micrograms ofpurified ES-62 in 50 microliters phosphate-buffered saline (PBS)subcutaneously upon day −2, day 0 and day 21 (ES-62 group). A secondgroup was additionally given ES-62 on days 22, 23 and 24 and every thirdday until the end of the study (ES-62 multi group) Control mice weresimply given PBS (PBS group). All mice were also given 200 micrograms ofcollagen (bovine, type II; Sigma, Poole, Dorset) in 100 microlitersFreunds complete adjuvant intradermally on day 0 and collagen in PBSintraperitoneally on day 21. A summary of the experimental protocol isshown in FIG. 3. In a separate therapeutic experiment performed aftercompletion of the work just referred to, ES-62 (2 mg) was not givenuntil 1 day after disease was established as being present and was thenadministered for fourteen consecutive days.

The incidence and severity of arthritis were measured daily up until day50. Severity scores were derived as follows: 0=normal, 1=erythema,2=erythema plus swelling, 3=extension/loss function, and total score=sumof four limbs. In the therapeutic study, measurements were undertakenduring the 14 days in which ES-62 was administered.

Lymph node cells were recovered at day 33 and day 50 (one day after endof ES-62 administrations for therapeutic study) and proliferation andcytokine responses to collagen measured. Briefly cells were cultured at2×10⁶ cells/ml for up to 96 h in medium [RPMI (Gibco/BRL, Paisley,Glasgow U.K.), supplemented with 2 mM L-glutamine, 100 IU/ml penicillin,100 mg/ml streptomycin, 25 mM HEPES buffer and 10% FCS (all Gibco/BRL)]at 37° C. in 5% CO₂. Cells were stimulated with graded concentrations ofcollagen. Proliferation assays were performed in triplicate in U-bottom96-well plates (Nunc, Roskile, Denmark) as described previously (Leung,B. P. et. al. (2000) J. Immunol. 164, 6495). Supernatants from paralleltriplicate cultures were stored at −70° C. until estimation of cytokinecontent by ELISA. Briefly, murine TNFα, IFNγ, (R&D Systems), IL-6, IL-10and IL-12 (p40+p70) (PharMingen, San Diego, Calif.) were assayed byELISA using paired antibodies according to the manufacturer'sinstructions. Lower limits of detection were as follows: IL-6, IL-12 andTNFα all at 10 pg/ml; IL-10 and IFNγ at 80 pg/ml.

Serum samples were recovered on day 50 (one day after end of ES-62administrations for therapeutic study) and antibody responses tocollagen measured by ELISA as described previously (Leung, B. P. et. al.(2000) J. Immunol. 164, 6495). Briefly, 96-well plates (Maxisorb, Nunc,Denmark) were coated with collagen (2 mg/ml in 0.1M NaH₂CO₃) overnightat 4° C., blocked and serial dilutions of sera were added. Bound IgG1/2awere detected with the aid of biotin-conjugated anti-mouse IgG1 or IgG2a(PharMingen) respectively.

Statistical analysis was performed using Minitab software for Macintosh.Clinical scores were analysed with the non-parametric Mann-Whitney Utest. Differences between cumulative incidences at a given time pointwere analysed by the chi-square contingency analysis. Cytokine andcollagen-specific IgG levels were compared using the Student's t-test.

On completion of the therapeutic study, mice had their hind paws removedand sections were prepared and stained with hematoxylin and eosin.

Balb/c or C57/B6 mice (groups of 5) were given 2 micrograms of purifiedES-62 in 100 microliters PBS subcutaneously upon day −2, day 0 and day 2(ES-62 group, Trial I). A second group of C57/B6 mice was additionallygiven ES-62 on days 4 and 6 (ES-62 group trial II) Control mice weresimply given PBS (PBS group). All mice were also given 5×10⁶ Leishmaniamajor parasites in 50 μl PBS subcutaneously in one footpad. Parasiteburden was assessed by the increment of footpad size (mm) in theinfected footpad relative to paired uninfected footpad as describedpreviously (Piedrafita D. et al. (1999) J Immunol 163:1467).

As shown in FIG. 4, the three-dose ES-62 protocol (ES-62 group)effectively reduced (statistically significant) the severity ofdeveloping arthritis (top panel). Initial studies indicate that theES-62 multi treatment (ES-62 multi-group) may be more effective than thethree dose ES-62 protocol (ES-62 group).

As shown in FIG. 5, the amelioration in severity of disease was matchedex vivo by suppression of antigen-induced cell proliferation in lymphnode cultures at day 33. This effect was entirely specific:proliferative responses to the non-specific mitogen, Concanavalin A werenot impaired.

As shown in FIG. 5, the amelioration in severity of disease was alsomatched ex vivo by suppression of production of the pro-inflammatorycytokines IL-6, TNFα and IFNγ in lymph node cultures at day 33.

Conversely as shown in FIG. 5, the production of the anti-inflammatorycytokine IL-10 was elevated.

As shown in FIG. 6, multi ES-62 treatment was effective in maintainingthe inhibition of both cell proliferation and inflammatory cytokineproduction (day 50).

As shown in FIG. 7, production of the TH-1 antibody, IgG2a was reducedby the multi-treatment (day 50).

As shown in FIG. 8, therapeutic treatment with ES-62 after the onset ofdisease was effective in preventing progression of established arthritisin the murine collagen model.

As shown in FIG. 9, the prevention of further development of disease wasmatched ex vivo by suppression of antigen-induced cell proliferation inlymph node cultures prepared one-day after completion of 14 days ofdaily ES-62 administration.

As shown in FIG. 9, the prevention of further development of disease wasalso matched ex vivo by suppression of production of thepro-inflammatory cytokines IL-6, TNFα and IFNγ in lymph node culturesobtained one-day after completion of 14 days of daily ES-62administration.

As shown in FIG. 10, production of the TH-1 antibody, IgG2a was reducedby the daily administration of ES-62 as detected one-day aftercompletion of 14 days of daily ES-62 administration.

As shown in FIG. 11, treatment with ES-62 in the therapeutic studyprevented the profound cartilage surface erosion and inflammationobserved in mice which simply received PBS.

As shown in FIG. 12, treatment with ES-62 in the therapeutic studyprevented the profound cartilage surface erosion and inflammationobserved in mice which simply received PBS.

TNF production by synovial tissues is an important surrogate forpro-pathogenic pathways in vivo. FIG. 13 shows that ES-62 inhibits TNFαproduction from human tissues in vitro. Two single cell suspensions(i.e. A and B) prepared from RA synovial membrane or synovial fluid werepre-treated with ES-62 and subsequently stimulated with LPS (1000 ng/mlor 100 ng/ml) for 24 hours to optimize TNFα release in vitro. TNFαrelease into culture supernatants was measured by ELISA. Data aremean±SEM of triplicate cultures and are representative of three similarexperiments.

FIG. 13 shows that ES-62 inhibits pro-inflammatory cytokine productionfrom human synovial membrane and fluid cells.

CONCLUSIONS AND IMPLICATIONS

The data presented clearly indicate that the 3-dose ES-62 protocolsignificantly reduces severity of collagen-induced arthritis in mice.This reduction was associated with loss of the normal pro-inflammatoryresponse to collagen and the development of an anti-inflammatoryphenotype. This change was caused by a reversal in T-cell polarity—fromTH-1 to TH-2 (as witnessed by changes in proliferation and cytokineproduction). Of additional interest, the effect on disease severity, andupon pro-inflammatory cytokine production ex vivo, was maintained byfurther administration of ES-62 given every three days. This stronglysuggested that ES-62 has activity in suppressing ongoing arthritis. Theresults of the final experiment carried out—the therapeutic study, wereentirely consistent with this. In this experiment, where mice weretreated with ES-62 after the onset of pathology associated withcollagen-induced arthritis, ES-62 prevented the further progression ofdisease. Hence ES-62 could be of value in the treatment of humanarthritic disease. The presented data on effects of ES-62 on productionof TNFα in human synovial cultures are consistent with this.

Finally, ES-62 may be a selective anti-inflammatory agent as it does notappear to modulate all diseases associated with polarisation of the Thelper phenotype. For example, in mice, susceptibility to infection withLeishmania is associated with a TH2 phenotype (Balb/c) whereasresistance and clearance of parasite is associated with a TH1 phenotype.Indeed, disease is exacerbated in mice deficient in pro-inflammatorycytokines such as IL-12 and TNF-alpha. Thus if ES-62 were to mediate itseffects simply by being “anti-inflammatory” or reversing TH1 polarity,it would be expected to exacerbate disease in Balb/c (TH2) mice andrender C57/B6 (TH1) mice susceptible to infection. In contrast,treatment with ES-62 had no effect on either strain of mouse.

1. A method of treating an inflammatory disease in an animal in needthereof comprising administering to said animal a therapeuticallyeffective amount of ES-62, wherein the inflammatory disease is selectedfrom the group consisting of arthritis and chronic obstructive pulmonarydisease.
 2. The method of claim 1, wherein the disease is arthritis. 3.The method of claim 1, wherein the disease is rheumatoid arthritis.